Analytical detection and quantification of drugs of abuse, therapeutic drugs, animal drug residues and environmental contaminants such as pesticides and industrial chemicals is an important and widespread commercial practice, and can be laboratory or field-based. The analytical equipment used in the laboratory and field can be the same, but field-based methods are generally more restricted due to size and analytical capabilities. For example, although portable formats have been constructed, commonly used analytical methods for precise quantification are gas chromatography (GC) and liquid chromatography (LC) often linked to mass spectrometers (to give GC-MS and LC-MS, respectively), and are restricted to use in the laboratory. The major alternatives to the chromatographic-based methods make use of the interaction of molecules through chemical reactions or chemical complexation, the latter typified by antibody-ligand binding. The main advantages of these chemical ‘binding’ methods compared to chromatographic methods are their relative low cost, simplicity and portability, enabling use in the field.
Chloral hydrate, 2,2,2-trichloroethanediol, is used as a sedative, in anaesthesia and as a chemical precursor in synthetic organic chemistry. Its use to combat insomnia can lead to addiction and overdose (Gaullier et at 2001; Engelhart et at 1998). Chloral hydrate overdose is often detected by the Fujiwara reaction which detects the metabolite 2,2,2-trichloroethanol, followed by quantification using GC-MS. Its sedative properties have been exploited for illegal use; when mixed with alcohol it produces the tasteless and odorless “knockout drops” or “Mickey Finn”, historically used to facilitate robbery. Discussion of drug facilitated rape (DFR) routinely includes reference to chloral hydrate. However, definitive proof of its use in DFR is generally lacking which implies either that such use is rare or that there are issues with its detection. Chloral hydrate is rapidly metabolized (Breimer 1977), the main metabolites being trichloroethanol, trichloroethanol glucuronide (TCG) and trichloroacetic acid, with respective half-lives of 7-10 hours, 7-10 hours and approximately 4 days (PharmGKB: The Pharmacogenetics and Pharmacogenomics Knowledge Base, Chloral Hydrate, Accession ID:PA448925). During the twenty four hours following a single oral dose of chloral hydrate approximately 0.7% is excreted in the urine as trichloroethanol and 28% as TCG (Disposition of Toxic Drugs and Chemicals in Man, eighth edition). Analytical methods used to detect and quantify the metabolites of chloral hydrate include GC fitted with an ECD detector (Breimer 1977; Ikeda et al 1984) and the Fujiwara reaction plus GC-MS (Heller et al. 1992).
Chloral hydrate is a disinfection by-product (DBP) in the industrial chlorination of water, attributed to the reaction of chlorine with trace organic matter such as humic acid, and it has been classified as a possible human carcinogen by the US Environmental Protection Agency (EPA) and Health Canada. The US EPA has approved a method using Gas Chromatography-Electron Capture Detection (GC-ECD) for its quantification and Health Canada suggests a Tolerable Daily Intake (TDI) of 0.3 mg/d for the average person (Health Canada, Guidance on Chloral Hydrate in Drinking Water). Levels in drinking water have been measured at 1.2-8.4 ng/ml (maximum 23 ng/ml) in Canada, and 1.7-2.5 ng/ml (maximum 46 ng/ml) in the United States. Trichloroethylene is an industrial solvent used extensively in metal cleaning and degreasing and human exposure to the chemical can occur through contaminated air, drinking water or food. Upon inhalation or ingestion, the chemical is rapidly metabolized via two routes; glutathione conjugation is the minor route, oxidation by cytochrome P450 enzymes the major route. The principal oxidative route produces the epoxide which is rapidly transformed into chloral/chloral hydrate, followed by synthesis of trichloroethanol, and finally trichloroethanol glucuronide formation (L. H. Lash et al 2000). The glucuronide is found in the urine of humans or other mammals exposed to or administered trichloroethylene (Lash et al 2000; Stenner et al 1997, Kim and Ghanayem 2006) and has been linked to autoimmune disease.
To the inventors' know ledge, an immunoassay for the detection of chloral hydrate use and generation has never been described even though an immunoassay based test would be desirable. It is highly likely that this is due to the small size of the molecule and its metabolites, trichloroethanol, trichloroethanol glucuronide and trichloroacetic acid, which present difficulties for antibody development; for example, such small molecules present a small target epitope for an antibody, potentially compromising resultant antibody specificity and titre.
Thus, provision of an antibody-based assay for suspected chloral hydrate induced DFR, excessive chlorine in water and suspected trichloroethylene exposure would be a rapid and cheap alternative to current detection methods. Furthermore, an off-the shelf device, such as a dip-stick, would enable individuals or environmental monitoring agencies to detect chloral hydrate, chlorine and trichloroethylene indirectly by measurement of TCG in urine samples.